The aim of the research proposed in this application is to determine if specific processing of a homogeneous RNA species which is an analogue of the primary transcript of a Beta-globin gene will occur in extracts of Hela cells which have been reported to catalyze specific cleavage and/or splicing of primary transcripts. Results reported to date of specific in vitro processing of complex heterogeneous RNA fractions or endogenously synthesized adenoviral transcripts have not been unambiguous. This investigator believes that study of the action of alleged precursor -messenger RNA processing activities on a pure, well-characterized, and exogenously added RNA substrate will yield definitive results. The principal investigator has developed an in vitro transcription system which yokes the promotor specificity of SP6 RNA polymerase to the synthesis of arbitrarily large amounts of any RNA sequence of interest. The rabbit Beta4-globin gene will be subcloned adjacent to the SP6 RNA polymerase promoter site in the pBR322/SP6-RVII plasmid and transcribed in vitro with SP6 RNA polymerase. Subsequently, the Beta4-globin transcripts (radiolabelled when necessary) will be capped and polyadenylated in vitro as well. The final product obtained will, accordingly, resemble the authentic primary transcript of the gene and should be suitable for study of pre-messenger RNA processing activities. Specific cleavage and/or spliced products will be detected and analyzed by application of the following techniques: 1) gel electrophoresis (under denaturing conditions) and autoradiography, 2) nuclease protection ("Berk-Sharp') analyses, and 3) RNA blot-hybridization analyses. The long term objectives of this research project are to identify, purify, and biochemically characterize the macromolecular components required to conduct processing of eukaryotic pre-messenger RNA transcripts in vitro.